Methods of increasing plant yield

ABSTRACT

This invention provides novel methods for improving plant quality and yield in the presence of pathogens. The method increases the levels of pathogenesis-related proteins, such as PR1, phenylalanine ammonia lyase, or plant cell wall proteins such as hydroxyproline-rich glycoproteins, in a plant by contacting the plant with a plant systemic inducer and a reactive oxygen species wherein the amount of the reactive oxygen species is sufficient to increase the amount of the pathogenesis-related protein above the level induced by the plant systemic inducer in the absence of the reactive oxygen species. A preferred reactive oxygen species is peracetic acid; a preferred plant systemic inducer is salicylic acid.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/857,137,filed Sep. 17, 2001, now U.S. Pat. No. 6,582,961, which was a nationalstage filing under 35 U.S.C. § 371 of PCT application PCT/US99/28552,filed Dec. 2, 1999, which was a continuation in part of provisionalapplication 60/110,835, filed Dec. 3, 1998. All of these applicationsare hereby incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for increasing resistance of plants toplant pathogens. More specifically, this invention relates to thesurprising discovery that the application to plants of one or morereactive oxygen species and of one or more plant systemic inducers,either simultaneously or within a short time of each other, results inan increase in the level of pathogenesis-related proteins and ofsystemic acquired resistance in the plants over the effect of either onealone.

2. Background

Commercial cultivation of plants is a major part of the economy,encompassing not only crops grown for human food and animal feed, butalso those, like cotton, grown for fiber, and others, such as flowers,grown for beauty. The importance of plants to people and to the economycan hardly be overstated. Plants are, however, also subject to constantattack by insects, fungi, bacteria, viruses, nematodes, and otherpathogens. When pathogens find susceptible plants, these attacks canresult in the loss of yield and quality, and may result in the loss ofentire crops. These losses result in substantial economic harm to thegrowers and, in some areas of the world, contribute to famine.

Except for those farmers who practice organic farming, most attempts tocontrol pathogens involve the use of pesticides, such as fungicides andinsecticides. Many pesticides, however, have been withdrawn from themarket because they have undesirable environmental impacts, and manycurrently on the market are being scrutinized for their environmentalimpact and may be withdrawn in the future. In addition, few, if any,pesticides are effective against the full range of pests which mayattack a given crop from sowing to harvest to post-harvest storage.Thus, a number of different pesticides with different target organismsmay need to be applied. Each one must be applied at the correct time inthe growth of the plants to provide effective control of the targetorganism, each has its own requirements for handling and application,and each may require different, specialized equipment. Moreover, manypesticides are toxic or have toxic residues, and their use is thereforeoften restricted to certain windows of time before harvest, after whichthey cannot be used because of the potential danger to the consumer.During this window, the crop may be essentially unprotected, or yetanother agent, safer for use close to harvesting, may be needed. The useof traditional chemical agents therefore requires complicated planning,careful timing, and considerable effort.

While pesticides form the bulk of attempts by farmers to protect plantsfrom pathogenic attack, not all protection of plants against pathogenscomes from the application of pesticides. For decades, it has been knownthat plants also have a wide variety of structural and biochemicaldefenses against attack by pathogens. See, e.g., Agrios, G., PlantPathology, Academic Press, San Diego Calif. (3rd ed., 1988).

One of the biochemical defenses produced by plants in response to attackis induced resistance, in which plants which have been inoculated withbiological agents or pretreated with various chemicals developnonspecific resistance not only to the initial agent itself, but also toa variety of pathogenic agents, such as viruses, fungi, bacteria, andsome insects. Induced resistance usually commences in the area aroundthe initial inoculation, but over the course of a few days, may spreadto portions of the plant not inoculated, a phenomenon known as systemicinduced resistance, or as systemic acquired resistance (“SAR”).

A number of compounds, such as salicylic acid, can induce resistance.See, e.g., Klessig, D. and Malamy, J., Plant Mol. Biol., 26:1439–1458(1994); Raskin, I., Annu. Rev. Plant Physiol. Plant Mol. Biol.43:439–463 (1992). They can be used to induce local resistance, byinjection or spraying, or to induce SAR when absorbed, for example,through the roots. See generally, Agrios, supra, at Chapters 5 and 9.SAR develops some 7 days or more after exposure to the inoculant orchemical agent, and usually lasts for some 3 to 5 weeks. Id.

Because SAR protects plants against many different pests, increasing SARin crops could potentially decrease or even eliminate the need to applytoxic pesticides. Further, since SAR protects against a multitude ofpathogens, inducing SAR can eliminate the need for a number of separateagents which would otherwise be necessary to protect a crop, or reducethe amount of the separate agents which would otherwise be required.And, because the induction of SAR can essentially be performed byrepetitive action, use of this technique would demand far less effortfor the farmer than the currently required regimen of applying multipleagents, each with their own directions for handling, timing, amounts,concentrations, methods of application, and possible adverseinteractions.

One of the world's largest pharmaceutical companies has made an effortto develop the use of systemic inducers to protect crops in the field.To this end, it is bringing to market a systemic inducer,benzothiadiazole, under the trade name Actigard.™ But, the manufacturernow recommends that Actigard™ be used in combination with conventionalchemical agents in providing protection to crops. Thus, even a systemicinducer specifically selected, developed and tested for protection ofcrops has not eliminated the need for conventional pesticides evenduring the time the systemic inducer is being applied.

U.S. Pat. No. 5,607,856, teaches compositions and methods forsterilizing soil using oxygen radicals. The method involves contactingthe soil with a solution of an activated oxygen species, a water-solublephenolic complex extracted from a material such as humic material, adivalent cation, and a cation redox reducing agent.

What is needed in the art is a means of protecting a variety of crops,flowers, decorative and other plants in the field from pathogens moreeffectively, at lower cost, and with less effort than by the use ofpesticides and other traditional chemical agents. Moreover, what isneeded is a means of providing this protection with lower and lesslasting damage to the environment than caused by such conventionalagents. What is further needed is a means of increasing the protectionof crops from pathogens to levels above the levels obtainable by the useof systemic inducers alone, to more crops than can be protected by theuse of systemic inducers alone, and against a wider range of pathogens.The present invention addresses these and other needs.

SUMMARY OF THE INVENTION

This invention provides novel methods of protecting plants frompathogens. In one group of embodiments, the methods involve contactingthe foliage of a plant with a plant systemic inducer and a reactiveoxygen species, where the amount of the reactive oxygen species issufficient to increase the expression of phenylalanine ammonia lyase,glutathione S-transferase, hydroxyproline-rich glycoprotein, chalconesynthase, or pathogenesis-related proteins, in the plant above the levelwhich would be induced by the plant systemic inducer in the absence ofthe reactive oxygen species. The invention further provides a method ofcontacting a plant with a plant systemic inducer and a reactive oxygenspecies, where the amount of the systemic plant inducer is sufficient toincrease the expression of phenylalanine ammonia lyase, glutathioneS-transferase, hydroxyproline-rich glycoprotein, chalcone synthase, orpathogenesis-related proteins in the plant above the level which wouldbe induced by the reactive oxygen species in the absence of the plantsystemic inducer. The increase in pathogenesis-related proteins,phenylalanine ammonia lyase, glutathione S-transferase, orhydroxyproline-rich glycoprotein caused by contacting a plant with botha plant systemic inducer and a reactive oxygen species may be additivecompared to the level induced by either the systemic inducer in theabsence of the reactive oxygen species or by the reactive oxygen speciesin the absence of the systemic inducer, or they may be greater than anamount which would be additive.

The plant can be edible by humans or by animals, can be grown for fibercontent, such as cotton, can be used for or processed to become amedicine or medicament, or can be for decorative, ornamental, orrecreational use, such as turf grass, house plants, flowers, orChristmas trees.

The reactive oxygen species (“ROS”) can be peracetic acid, hydrogenperoxide, a hydroperoxide, a peroxide, or a phenolic hydroperoxide;ozone is not preferred as an ROS. The plant systemic inducer can besalicylic acid, jasmonic acid, isonicotinic acid, dichloroisonicotinicacid, benzothiadiazole, phosphorous acid, arachidonic acid, or cinnamicacid, can be derived from kelp or other seaweed, or can be a beneficialmicrobe. The systemic inducer can be humic acid, or can be used incombination with humic acid. The ROS and microbial plant systemicinducer can be administered about 24 hours of one another, whereas anROS and a chemical plant systemic inducer are preferably administeredwithin one hour of each other and even more preferably are administeredtogether in a mixture, either as, for example, a powder, or, morepreferably, a solution. The pathogenesis-related protein can be aproduct of any of the PR genes having or thought to have a role inprotecting plants from pathogens, such as the PR-1, PR-2, PR-3, PR-4 andPR-5 genes. The proteins induced can also be phenylalanine ammonialyase, chalcone synthase, or a hydroxyproline-rich glycoprotein or otherproteins related to strengthening of cell walls or plant defense.

The invention further provides a composition for foliar application toplants comprising a plant systemic inducer and a reactive oxygen specieswherein the amount of reactive oxygen species is sufficient to increasethe level of a natural plant product selected from the group consistingof: phenylalanine ammonia lyase; hydroxyproline-rich glycoproteins,glutathione S-transferase, chalcone synthase, and pathogenesis-relatedproteins to a level above the level induced by the plant systemicinducer in the absence of the reactive oxygen species. The compositioncan further comprise an aqueous solution and detergents, chelatingagents or sequestering agents.

In another group of embodiments, the invention provides methods ofprotecting a plant by contacting one or more roots of a plant with aplant systemic inducer and a reactive oxygen species, where the amountof the reactive oxygen species (ROS) is sufficient to increase theexpression of phenylalanine ammonia lyase, glutathione S-transferase,hydroxyproline-rich glycoprotein, chalcone synthase, orpathogenesis-related proteins, in the plant above the level which wouldbe induced by the plant systemic inducer in the absence of the reactiveoxygen species, provided that the composition does not comprise anexogenous agent selected from the group comprising a cation redoxreducing agent and a divalent cation having redox potential in amountssufficient to reduce the levels of microorganisms in soil around theroots by 40% or more. The invention further provides a method ofcontacting one or more roots of a plant with a plant systemic inducerand a reactive oxygen species, where the amount of the systemic plantinducer is sufficient to increase the expression of phenylalanineammonia lyase, glutathione S-transferase, hydroxyproline-richglycoprotein, chalcone synthase, or pathogenesis-related proteins in theplant above the level which would be induced by the reactive oxygenspecies in the absence of the plant systemic inducer, provided that thecomposition does not comprise an agent selected from the groupcomprising a cation redox reducing agent and a divalent cation havingredox potential in amounts sufficient to reduce the levels ofmicroorganisms in soil around the roots by 40% or more. The systemicinducer can be, or can be mixed with, humic acid.

The invention further provides compositions for soil application toplants comprising a plant systemic inducer and a reactive oxygen specieswherein the amount of reactive oxygen species is sufficient to increasethe level of a natural plant product selected from the group consistingof: phenylalanine ammonia lyase; hydroxyproline-rich glycoproteins,glutathione S-transferase, and pathogenesis-related proteins to a levelabove the level induced by the plant systemic inducer in the absence ofthe reactive oxygen species, provided that the compositions do notcomprise an agent selected from the group comprising a cation redoxreducing agent and a divalent cation having redox potential in amountssufficient to reduce the levels of microorganisms in soil around theroots by 40% or more. The systemic inducer can be, or can be mixed with,humic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a northern blot of RNA probed for expression of PR-1a protein.RNA was extracted from 14 day-old red kidney bean plants 24 hours afterleaves of the plants were sprayed until wet with one of five treatments.Lane 1: treatment with 3,500 ppm of peracetic acid (“PAA”). Lane 2:treatment with 10,000 ppm of PAA. Lane 3: treatment with 3,500 ppm ofPAA plus 200 ppm of salicylic acid/humic acid mixture (see Example 1;hereafter “inducer mixture”). Lane 4: treatment with 10,000 ppm of PAAand 200 ppm of inducer mixture. Lane 5: treatment with 200 ppm inducermixture, without PAA.

FIG. 2 is a northern blot of RNA probed for expression of phenylalanineammonia lyase (“PAL”). RNA was extracted from 14 day-old red kidney beanplants 24 hours after leaves of the plants were sprayed until wet withone of four treatments. Lane 1: water-only control. Lane 2: treatmentwith 3,500 ppm of PAA. Lane 3: treatment with 10,000 ppm of PAA. Lane 4:treatment with 3,500 ppm of PAA plus 200 ppm of inducer mixture.

FIG. 3 is a northern blot of RNA probed for expression ofhydroxyproline-rich glycoproteins (“HPRG” or “HYP”). RNA was extractedfrom 14 day-old red kidney bean plants 24 hours after leaves of theplants were sprayed until wet with one of five treatments. Lane 1:water-only control. Lane 2: treatment with 3,500 ppm of PAA. Lane 3:treatment with 10,000 ppm of PAA. Lane 4: treatment with 3,500 ppm ofPAA plus 200 ppm of inducer mixture. Lane 5: treatment with 10,000 ppmof PAA plus 200 ppm of inducer mixture. Lane 6: treatment with 200 ppmof inducer mixture, without presence of PAA.

FIG. 4 is a chart setting forth the results of a field trial of theeffect of applying a ROS/inducer mixture on levels of powdery mildewinfection of seedless table grapes. Plants were sprayed with an airblast sprayer with 200 gallons of water at a rate of 32 ounces per acre.The spray contained a mixture of PAA at a concentration of 1250 ppm andinducer mixture at a concentration of 125 ppm. Each replicate contained88 plants, and a total of 16 replicates were treated. Level 1 mildewindicates that fewer than 3 young grapes (called “berries”) per plantare infected with mildew. Diamonds: Percentage of plants in eachreplicate with Level 1 mildew prior to treatment. Squares: Percentage ofplants in each replicate with Level 1 mildew following treatment.

DETAILED DESCRIPTION

I. Introduction

This invention arises from the surprising discovery that the applicationto plants of one or more reactive oxygen species (“ROS”) and of one ormore systemic inducers, such as salicylic acid, either simultaneously orwithin a short time of each other, results in an increase in the levelof transcription of hydroxyproline-rich glycoproteins (“HRGP”),phenylalanine ammonia lyase (“PAL”), chalcone synthase, peroxidase (PAL,chalcone synthase, and peroxidase are sometimes referred to as a plant's“phenolic defenses”), glutathione S-transferase, or pathogenesis-relatedproteins (“PR proteins”), and of SAR in the plants over the effect ofeither one alone. The increase is additive of the effects of ROS or of asystemic inducer alone and, even more surprisingly, may be synergistic.

This result could not have been predicted. What was known was that ROSand systemic inducers each cause a set of genes to be expressed, butsome of the genes were the same and some were different for each classof agent. Since the mechanisms of signaling within the plant cellrelated to SAR are not yet elucidated, there was no way to predictwhether applying both agents at or around the same time would resultsimply in the same amount of induction of SAR as applying one of theagents, or whether the amount induced would be greater than or less thanapplying one of these agents alone.

We have discovered, for example, that contacting plants with both agentsresults in increases in levels of transcription of PR proteins over thatexpected. “The expression of many of the well characterized PR genes(e.g. PR-1 through PR-5) in tobacco has been correlated with resistanceto a large variety of viral, bacterial and fungal pathogens. As aresult, expression of PR genes is often used as a marker for inductionof disease resistance.” Klessig and Malamy, supra, at 1441. See also,e.g., Bowles, D., Annu Rev. Biochem 59:873–907 (1990); Carr, J. andKlessig, D., The pathogenesis-related proteins of plants. In Setlow, J.(ed.) Genetic Engineering Principles and Methods, vol. 11, pages 65–109.Plenum Press, New York (1989); Dixon, R., et al., Adv Genet. 28:165–234(1990); Ward, E., et al., Plant Cell 3:1085–1094 (1991); Ye, X., et al,Plant Sci., 84:1–9 (1992); Woloshuk, C., et al, Plant Cell 3:619–628(1991); Alexander, D., et al, Proc Natl Acad Sci USA 90:7327–7331(1993); Bol, J., et al, Annu Rev Phytopath 28:113–138 (1990); Ward, etal., Plant Cell 3:1085–1094 (1991); Niederman, et al., Plant Physiol108:17–21 (1995); Lawton, et al., Plant J. 10:71–82 (1996); andFriedrich, L., et al., Plant J. 10:61–70 (1996).

We have shown that, in addition to the PR proteins, such as PR-1a, thetranscription of other enzymes and proteins, such as PAL, chalconesynthase, and HRGP, increase in response to contacting plants with ROSand increase even more in response to contacting plants with bothsystemic plant inducers and ROS. The increases are greater than thatinduced by either class of agent alone, and may, indeed, be synergistic.And, while SAR is considered to take a week or more to develop, we havedemonstrated that genes encoding the enzymes noted above are inducedwithin 24 hours of contacting plants with these compositions. It isrecognized in the art that the transcription of these genes is a markerfor SAR.

In addition to these discoveries, a water soluble phenolic complex knownas humic acid commercially sold as a fertilizer also acts as a systemicinducer. Contacting plants with humic acid induces changes in many ofthe same enzymes as those increased in response to previously knownsystemic plant inducers and that the levels of these enzymes increaseeven more when plants are contacted with both humic acid and ROS. Aswith previously known systemic inducers, the increases are greater thanthat induced by either class of agent alone, and may, indeed, besynergistic. Moreover, adding humic acid to a mixture of a previouslyknown systemic inducer, such as salicylic acid, and an ROS results inyet a further improvement in the level of enzymes considered to be partof a plant's defense mechanisms.

The increases we have shown in the levels of PAL, HRGP, chalconesynthase, PR proteins or other marker proteins and enzymes uponapplication of the two types of agents is also mirrored in the field bylevels of protection against pathogens comparable to or greater than theprotection provided by conventional chemical agents directed againstspecific types of pathogens. As shown in the Examples, for instance, theapplication of a mixture of ROS and of a mixture of the systemic inducersalicylic acid and humic acid (hereafter “ROS/inducer mixture”) to cropssuch as grapes, lettuce, tomatoes, carrots, and citrus fruit in fieldtrials resulted in rates of infestation and amounts of damage fromfungi, insects, and nematodes comparable to or lower than the rates ofinfestation and amounts of damage from the same pests to crops protectedby conventional agents directed against those specific types ofpathogens.

Moreover, this increased, and possibly synergistic effect allows the ROSand systemic inducers to be applied at rates which render themcommercially viable compared to the pesticides, fungicides, or otheragents which would otherwise be needed. Further, since ROS and systemicinducers are far less toxic to handle and apply than most conventionalpesticides, use of the invention reduces the exposure of farmers andother agricultural workers to toxic chemicals they may be poorly trainedor equipped to use. And, since SAR can be raised to protective levels bytreatments (such as by the preferred embodiment of a mixture of an ROSand one or more SAR inducers) which are both relatively non-toxic toproduce and much less environmentally damaging in use than manyconventional pesticides and other chemical agents, the inventionprovides crops with meaningful protection from pathogens while sharplyreducing the cost to the environment of that protection. Finally, asnoted above, the protection has been demonstrated with respect to avariety of divergent plants and against a range of pathogens. Theinvention is therefore a significant and substantial improvement overthe application of a systemic inducer alone.

It should be noted that increases in SAR in some cases may increase aplant's susceptibility to certain pathogens. But we predicted that thiseffect would be far outweighed by the increase in protection against amuch wider range of pathogens than those to which a plant might becomemore susceptible, and that this effect would result in improved cropyield and quality. This prediction has been confirmed by the increasesin crop yield and quality demonstrated in the field trials reportedherein. Accordingly, the application of a systemic inducer and an ROShas been shown to have a positive effect in protecting crops in thefield.

II. Definitions

The term “combining” as used herein refers to the mingling of two ormore liquid, solid or aerosolized components before, during or aftercontact to plants.

The phrase “increase the level above the level induced by the plantsystemic inducer in the absence of the reactive oxygen species”specifies a level of natural plant product. The level from which changeis measured is the level of the natural plant product of interest thatis induced in a plant by contact with a plant systemic inducer when areactive oxygen species is absent. Therefore, the phrase quoted aboverefers to any concentration of a natural plant product that is abovethis level. Similarly, the phrase “increase the level above the levelinduced by the reactive oxygen species in the absence of the plantsystemic inducer” specifies a level of natural plant product. The levelfrom which change is measured is the level of the natural plant productof interest that is induced in a plant by contact with a reactive oxygenspecies when a plant systemic inducer is absent. Therefore, the phrasequoted above refers to any concentration of a natural plant product thatis above this level.

The terms “administering” and “contacting” plants with a chemical orcompound, as used herein, generally comprehend causing the plant to comeinto proximity with an exogenous liquid or solid (such as a powder) formof the chemical or compound. They do not comprehend the injection ofcompounds or chemicals into individual leaves or into individual plants.

The terms “hydroxyproline-rich glycoproteins” or “HRGPs,” as usedherein, refer to glycosylated proteins found in plant cell walls andassociated with cell wall strengthening. The HRPGs are characterized bythe presence of the motif: Ser-(Hyp)₄-Tyr, wherein “Hyp” ishydroxyproline.

The terms “pathogenesis-related protein,” or “PR proteins,” as usedherein, refer to any of a number of families of proteins whose synthesisis considered in the art to be induced in response to, contact with, orinfection by, a pathogen. They are considered to be encoded by, forexample, the PR-1 to PR-5 genes. The expression of pathogenesis-relatedproteins may also be induced or increased in a plant by contacting theplant with plant systemic inducers, such as salicylic acid. Thefunctions of many PR proteins are known. For example, the PR-2 genesencode hydrolytic β-1,3 glucanases, while the PR-3 family encodeschitanases. Other pathogenesis-related proteins, however, such as thoseexpressed by the PR-1 gene group, have functions which have yet to bedefined. Expression of PR-1a is, however, considered by those of skillin the art to be associated with, and a marker for, systemic acquiredresistance.

The term “natural plant product,” as used herein, refers to an enzyme orstructural protein endogenously produced by a plant. Examples of suchcomponents include phenylalanine ammonia lyase, hydroxyproline-richglycoproteins, and pathogenesis-related proteins.

As used herein, the term “pathogen resistance” refers to the ability ofa plant to lessen the development of disease symptoms after exposure toa plant, insect or microbe.

The term “phenylalanine ammonia lyase” refers to an enzyme thatcatalyzes the conversion of phenylalanine into cinnamic acid. Theenzyme, EC number 4.3.1.5., is involved in the formation of many classesof phenolic compounds involved in plant defense.

The term “glutathione S-transferase” refers to any of a family ofenzymes which transfer glutathione in the course of any of variousfunctions, including many involved in stress responses in plant cells.The enzymes play a role in oxidative stress, herbicide resistance andheavy metal tolerance. See, e.g., Neufield T, et al., J. Biol Chem378:199–205 (1997); Chen W, et al., Plant Journal 10:955–966 (1996);Levine, A., et al., Cell 79:583–593 (1994).

The term “chalcone synthase” refers to an enzyme which is involved inthe synthesis of more complex phenolic structures of chalcones essentialfor flavonoid synthesis. Flavonoids represent a major class of plantsecondary metabolites that are important in plant survival. They areknown to play a role in a wide range of plant functions, includingpathogen protection. The enzyme, EC number 2.3.1.74, catalyzes thecondensation of three molecules of malonyl-CoA with one molecule of4-coumaroyl-CoA to produce chalcone. See, e.g., Hahlbrock K, Flavonoids.In Stumpf P K, Conn E E, eds, Biochemistry of Plants, Academic Press,New York (1981), pp 425–456; Niesbach-Klosgen U. et al., J Mol Evol26:213–225 (1987).

The terms “plant systemic inducer,” “systemic inducer of resistance,”and “systemic inducer” are used herein as synonyms and as used hereinrefer to chemical or biological agents that induce pathogen resistanceafter a plant is contacted with a plant systemic inducer. Examples ofchemical plant systemic inducers include, inter alia, salicylic acid,jasmonic acid, isonicotinic acid, dichloroisonicotinic acid, phosphorousacid, and cinnamic acid, chitosan, humic acid, and beta-1,3 glucans andother mixed glucans.

One skilled in the art will recognize that biological plant systemicinducers include, inter alia, bacteria, viruses, fungi, and nematodes.Kelp, a form of seaweed, and some other seaweeds, are rich sources ofbeta glucans and can be used as systemic inducers in the compositionsand methods of the invention. Unlike bacteria, viruses, and most othermicrobiological agents, kelp and other seaweeds are sold commercially asfertilizers and are available as liquid extracts or as dried powders. Inthis regard, they more resemble agricultural chemicals and for ease ofdiscussion, will therefore be treated as chemical inducers herein unlessotherwise indicated. Finally, as noted in the Introduction, humic acid,a component found in the humus portion of some soils, also acts as asystemic inducer. Thus, the term “systemic inducer” can, whereappropriate, include reference to humic acid. In preferred embodiments,we have found good results by including humic acid along with othersystemic inducers in the compositions and methods of the invention.Accordingly, the addition of humic acid to other systemic inducers willgenerally be specifically denoted herein.

The phrase “reactive oxygen species” (abbreviated herein as “ROS”)describes oxygenated compounds which serve as a source of oxygenatedradicals. The term is considered to be synonymous with “activated oxygenspecies.” These compounds include, inter alia, peracetic acid, sodiumperoxide, potassium oxide, potassium peroxide, calcium peroxide,magnesium peroxide, urea peroxide, hydrogen peroxide (H₂O₂),hydroperoxides (ROOH), peroxides (ROOR), and superoxides, where R is analkane, alkene or alkyne, branched or unbranched, and of between 1 and12 carbons and Ar is an aromatic ring, usually of 6 carbons, or acombination of such rings. As used herein, the term “reactive oxygenspecies” excludes the gas ozone.

Humus is the major organic component of soil. “Humic acid” is a phenoliccomplex which is a component of humus. Commercially, humic acid isgenerally extracted from what is described as a salt-free deposit ofhighly oxidized carbon known as “Leonardite.” Extraction of humic acidfrom Leonardite is described in detail in U.S. Pat. No. 5,607,856.

“Peracetic acid” is a reactive oxygen species which is made by reactingglacial acetic acid with hydrogen peroxide. Since this reaction does notgo to completion but instead results in a equilibrium being reached, atany point in time all three chemical species, peracetic acid, aceticacid, and hydrogen peroxide, will exist. Use of the term “peraceticacid” herein therefore encompasses mixtures of these three chemicalspecies.

The phrase “field capacity” refers to the percent water remaining in thesoil two to three days after having been saturated and after freedrainage has practically ceased.

The term “foliar application” refers to the application of substances tothe foliage, or above-ground portions, of plants, and especiallyapplication to the leaves of the plants. It is understood in the artthat incidental amounts of substances used in foliar applications mayfilter to or contact the soil, but not in quantities which will permitpenetration of the soil and significant contacting of the plant's rootscompared to the amount contacting the leaves and other above-groundstructures.

The term “soil application” refers to the application of a substance tothe soil around a plant, where the intent is either to affect the soildirectly or to place the roots of the plant in contact with thesubstance. Generally, substances applied through a soil application willnot contact the foliage, but it is possible that incidental amounts ofsubstances used in soil applications may contact the foliage inquantities which will not significant compared to the amount contactingthe roots and other below-ground structures.

The term “crop,” as used herein usually refers to plants raised infields in an agricultural setting, and includes, along with tomatoes,grapes and other plants intended for human or animal consumption, plantsintended for use as fibers, plants to be used as or processed intomedicaments, plants grown for fragrance, flowers, herbs, and decorative,recreational, and ornamental plants. In this context, the term includestree farms, such as those growing conifers to be used as Christmastrees, and grasses grown for use as turf. The term can also encompass,in context, plants grown in greenhouses.

References to an “ROS/inducer mixture” mean a mixture of one or morereactive oxygen species (such as peracetic acid) and one or more plantsystemic inducers.

Unless otherwise specified, references herein to “parts per million” (or“ppm”) used in reference to a mixture of an ROS (which is usually in asolution with other ingredients, such as sequestering agents andsurfactants) and a systemic inducer (which is also usually in a solutionwith chelating agents, surfactants, or other ingredients, which inpreferred embodiments will contain humic acid), refers to theconcentration of the solution containing the ROS component, as will bemade clearer in the discussion below. The term is also used herein inreference to a mixture of an ROS (which is also usually in a solutionwith chelating agents, surfactants, or other ingredients, which inpreferred embodiments will contain a systemic inducer) and humic acid.

Unless otherwise specified, reference herein to a particularconcentration of an ROS/inducer mixture or of an ROS/humic acid mixtureis of the concentration of the ROS portion of the mixture (including anyadditives), but implies the presence of a systemic inducer (includingany additives) or of humic acid (including any additives), respectively,at a concentration about one-tenth that stated for the ROS portion ofthe mixture. That is, if an ROS/inducer mixture is stated to be appliedat 2500 ppm, the ROS portion of the mixture (including relatively smallamounts of any additives, such as surfactants, chelating agents or otheringredients noted herein) is 2500 ppm, and a systemic inducer is presentat about 250 ppm (including relatively small amounts of any surfactants,chelating agents or other ingredients which may be added to theinducer). Typically, the ROS/inducer mixture will be in an aqueoussolution, but in some formulations, can be applied as a mixture of dryingredients which will be wetted after application.

The phrase “cation redox reducing agent” is any reducing agent thatdonates electrons to a cation that has participated in the generation ofan oxygenated radical. In certain instances, the cation is oxidized backto its active species, thus acting as a “free radical pump,” capable ofagain generating oxygenated radical species.

The phrase “divalent cation having redox potential” is any divalentcation that can accept additional electrons.

As used herein, the phrase “reducing the level of microorganism by 40%”means that there are at least 40% fewer microorganisms present in asample of soil contacted with a composition than there are in a controlsample which has not been contacted with the same composition. Thenumber of microorganisms present can be determined by any of a number ofassays known in the art, such as by plating out samples on agar platesand quantitating the resulting colonies.

The term “exogenous,” with respect to the presence of a cation redoxreducing agent or divalent cation having redox potential, means that theagent or divalent cation is added to a composition to raise itsconcentration over that which normally or naturally be present. It doesnot include trace amounts which might naturally be present in soil ormanufactured compositions (such as humic acid, which is typicallyextracted from Leonardite), or which might be added as an incidentaleffect of normal processing. For example, minor amounts of the divalentcations manganese or copper might leach into a composition stored in acontainer made of those materials, but such contaminants would not beconsidered the addition of an exogenous divalent cation for purposes ofthis invention.

III. ROS/Inducer Mixtures

a. Reactive Oxygen Species and Methods of Use

As noted, the method of the invention involves the use of a reactiveoxygen species (“ROS”) and one or more systemic inducers. A number ofROS can be used in the methods of the invention. Many ROS compounds arecommercially available. In general, ROS with higher active oxygenquotients are preferred. Preferred ROS for use in the invention include,for example, peracetic acid, hydrogen peroxide, calcium peroxide, sodiumpercarbonate, and urea peroxide. Less preferred are sodium peroxide andmagnesium hydroxide.

Peracetic acid is the ROS particularly preferred for use in theinvention. It is much more stable than hydrogen peroxide and has ahigher active oxygen quotient. It is also commercially available from anumber of sources, including FMC Corporation (Chemical Products Group,Philadelphia. Pa.), Solvay Interox (Warrington, United Kingdom) andDegussa Corporation (Ridgefield Park, N.J.). It is desirable that theperacetic acid used be shelf-stable, although non-shelf stable acid canbe used if it will be used before substantial loss of active oxygenoccurs. The most commonly available form of peracetic acid is made usingsulfuric acid. The use of peracetic acid made in this manner is notpreferred, since any residue of sulfuric acid which may remain in theperacetic acid will be phytotoxic. Accordingly, this form of peraceticacid should not be used unless any residue of sulfuric acid has beenreduced to levels which are not phytotoxic. Peracetic acid made by othermethods is preferred. Peracetic acid should be handled in stainlesssteel or plastic approved for the purpose to reduce contamination anddecomposition. Typically, the peracetic acid is mixed to a concentrationof 5% (w/v).

One significant problem in using ROS is heavy metal contamination, whichcauses premature decomposition of the ROS and, hence, a reduction in itseffectiveness. To reduce the amount of contamination by heavy metals,small amounts of chelating or sequestering agents, such astetrapotassium pyrophosphate, can be added to sequester heavy metalions. Since dust can blow into the mixture as it is being prepared evenin applications where it would not appear that heavy metal contaminationwould be a problem, it is desirable that ROS used in the invention havea small amount (0.05% by weight), of tetrapotassium pyrophosphate oranother chelating or sequestering agent present as a precaution. The useof polyethylene, plastic tanks, stainless steel tanks, and polyethylene,plastic, or stainless steel lines, is preferred in handling peraceticacid.

The amount of heavy metal contamination can also vary by the means ofapplication, since some farm equipment, for example, such as metallicsprayers and irrigation equipment, can be expected to have a higherlevel of heavy metal contamination than, for example, a rubber hose. Ahigher level of chelating or sequestering agent should be used insituations where the application equipment may itself have heavy metalcontamination. Conversely, many ROS start decomposing if the level of achelating or sequestering agent reaches too high a level. To reduce thisproblem, higher levels of chelating or sequestering agent are added tothe systemic inducer, which is not mixed with the ROS until shortly orimmediately before use, so that there will not be sufficient time beforeapplication to the plants for a substantial amount of decomposition tooccur, while the chelating or sequestering agent is still present toprotect the ROS while it is exposed to the possible contamination.Conveniently, where the ROS and the systemic inducer are prepared wellin advance of use, they are packaged in separate containers, onecontaining the ROS and the other the systemic inducer and thesequestering agent.

Peracetic acid is usually used in an aqueous solution of a desiredconcentration. Conveniently, it is made in a 5% solution, to whichsequestering agents, surfactants, and other agents can be added. Thecalculations herein concerning concentrations of ROS or of ROS/inducermixture were made using a 5% solution of peracetic acid solution, withthe sequestering agents and other additives noted above. The solutionwas considered to represent 100% for purposes of calculating parts permillion. Other concentrations of peracetic acid or other ROS can ofcourse be used, with appropriate adjustment in the calculations todetermine the parts per million resulting from any dilution. Forexample, if a 15% solution of peracetic acid is used, than only onethird the amount of peracetic acid would be needed to supply the samenumber of parts per million. It is well within the ability of thepractitioner to calculate the ROS present in parts per million for anygiven starting concentration.

Although peracetic acid is used in an aqueous solution, othercompositions exist which form reactive oxygen species upon the additionof water. These compositions can be used, for example, to reducetransportation and handling costs associated with the ROS. Compoundsuseful in this regard include calcium hydroxide, sodium percarbonate,and potassium permanganate, with sodium percarbonate and potassiumpermanganate being less preferred. Typically, the composition is appliedto a field and the field is then watered. These compounds are applied atrates which produce after watering concentrations equivalent to thosediscussed herein for the aqueous solutions of peracetic acid.

b. Systemic Inducers and Method of Use

A variety of systemic inducers can be used with the ROS. Preferably, theplant systemic inducer used will be either a biological inducer, such asa beneficial microbe, or a chemical systemic inducer. Biologicalcomponents, such as glycoproteins, can now be routinely manufacturedthrough recombinant techniques.

While biological inducers such as microbes can be used, it is usuallymore convenient to use chemicals systemic inducers, which can besynthesized in bulk at reasonable cost. Preferred systemic inducers aresalicylic acid, jasmonic acid, isonicotinic acid, chitosan, beta-1,3,glucan, other mixed glucans, dichloroisonicotinic acid, Messenger™ (EDENBioscience Corp., Bothell, Wash.), and Actigard.™ In some embodiments,the systemic inducer can be a glucan-containing kelp, such asAscophyllum nodosum and Laminaria digitata, or other seaweeds. Suchkelps and other seaweeds are commercially available as fertilizer orplant nutrient supplements from a number of sources, such as NorthAmerican Kelp (Waldoboro, Me.), Thorvin, Inc. (New Castle, Va.),American Kelp Corp. (San Diego, Calif.), Agrikelp (Colburne, Ontario,Canada), and Maxicrop USA, Inc. (Elk Grove Village, Ill.). Humic acidcan also be used as a systemic inducer, or as a one component of amixture containing at least one other systemic inducer.

One particularly preferred systemic inducer is salicylic acid. Thiscompound is available commercially in solid form. Typically, the solidform is mixed with a base to create a salt, which is readilysolubilized. While caustic soda or other high pH substances can be used,caustic potash is preferred as the base since the potassium in thecaustic potash is a plant nutrient and is therefore compatible with theuse of the resulting mixture as an agricultural product. After formingthe salt, a small amount (˜1%) of 80% phosphoric acid is added to bufferthe solution since it tends to still have a high pH. Phosphoric acidshould be added until the pH is reduced to about 8 (other inducers mayhave optimal activity at other pHs, which can be readily determined bysimply applying the mixtures at different pHs to plants and thenassaying for expression of SAR-related genes as taught in the Examples).Small amounts (2.5%) of surfactants may also be added to help absorptionof the mixture by plant roots, leaves, and other plant surfaces.Surfactants are commonly used as wetting agents; commercially availableagents suitable for use in the invention include Triton H-66™ andTergitol 15 S.™ In a preferred embodiment, 2.5% of each of thesesurfactants is added to the systemic inducer mixture. Other surfactantscan be used. The surfactants should be selected for compatibility withperacetic acid or the particular ROS which will be employed. Forexample, one can determine the active oxygen quotient of the ROS byindustry standard methods, proceed to mix the ROS with the desiredsurfactant, and retest the active oxygen quotient. A loss of more than1% is generally considered to mean the two agents are incompatible.

In some embodiments, two or more systemic inducers may be administeredto increase the robustness of the response. Typically, the totalconcentration of the inducer portion of the ROS/inducer mixture willremain the same, but will be divided between or among the inducersselected for the application in question.

In preferred embodiments, humic acid is added to be 0.1% to 50% of thetotal systemic inducer present, although humic acid can also be used asthe major species of inducer present, constituting more than 50% of thetotal systemic inducer present, and can if desired be the only systemicinducer used. We have found particularly good results mixing salicylicacid and humic acid in a 1:1 ratio and adding this mixture to the ROS toform the ROS/systemic inducer mixture. It should be noted that thestandard grade of humic acid has a solubility of 12%, but higherpercentages of solubility may be possible for some formulations. Thepercentages stated for humic acid as a component of a systemic inducermixture are for a 12% solution, and the percentages can be adjusted asappropriate if the solution used is of a higher or lower percentage.

In the most preferred embodiment, the systemic inducer is administeredat about one-tenth the concentration of the ROS. Conveniently, thesystemic inducer is mixed in a 10% solution. The ROS and chemicalinducer can then be applied at equal volumes to maintain the desired 10to 1 proportion. For example, one gallon of the ROS solution (such asthe 5% solution of peracetic acid noted in the preceding section asconsidered to be a 100% solution for purposes of these calculations) canbe mixed in a tank with one gallon of the 10% systemic inducer solution.The resulting mixture can then be diluted to any desired level of partsper million. It should be noted again that for purposes of calculatingparts per million, we consider only the ROS solution component. Theparts per million of the systemic inducer portion is implied at about10% of the concentration of the ROS, but is not separately calculated orconsidered in the ppm calculation. Thus, a calculation of “2500 ppm”refers only to the ROS component (with all of its surfactants and thelike), without including the 250 ppm of systemic inducer which would beadded by the systemic inducer portion of the ROS/systemic inducermixture.

Beneficial microbes can also be used as systemic inducers. A number ofmicrobes are known to act as systemic inducers, and usually, the microbechosen is not pathogenic to the plant to which it is to be applied; forexample, it may be avirulent or a microbe to which the plant in questionis resistant, or, preferably, a saprophyte. E.g., Klessig and Malamy,supra, at 1440. Preferably, the microbe should be nonpathogenic to theplant and one which improves the plant's growth, yield and quality. Forexample, species of bacteria such as Bacillus, Serratia, and Pseudomonasand fungi such as Trichoderma are known to act as systemic inducers. Itshould be noted, however, that the effects of any particular bacterialspecies can vary by soil type, the time of year, and the particular cropto which the organism is to be applied. Accordingly, the practitionerwill usually first test the organism on the crop in a small field toascertain whether a particular microbe is beneficial to the crop inquestion, and will examine the growth, yield and quality against anontreated or mock-treated crop, until he has identified organismsbeneficial in his fields, on the crop in question.

Microbial inducers are usually applied live at between around 10³ and10¹⁰ colony forming units (CFU) per milliliter. The microbial inducerscan be applied by spray or by irrigation. Typically, between about ¼gallon and 75 gallons of microbial inducer at this concentration isapplied per acre. The microbial inducer can be applied in any convenientamount of water so long as the desired amount of microbial inducer (forexample, 5 gallons of microbial inducer culture of between about 10³ and10¹⁰ CFU per ml) is applied. In a more preferred embodiment, about ½gallon to about 50 gallons per acre is applied, and in a still morepreferred embodiment, about ¾ gallon to about 25 gallons per acre isapplied. Even more preferred, about ¾ gallon to about 10 gallons isapplied per acre. In the most preferred embodiment, about 1 gallon toabout 5 gallons are applied per acre.

c. Administration of ROS and Inducers

Usually, the ROS and a chemical systemic inducer will be administeredwithin 36 hours of one another. More preferably, the ROS will beadministered within 24 hours of administration of the systemic inducer.Even more preferably, the ROS will be administered within 12 hours orless of administration of the systemic inducer. Still more preferably,the ROS and a chemical systemic inducer will be administered withinabout an hour of each other. Most preferably, the two agents will beapplied together as a mixture in an aqueous solution. The methods of theinvention do not contemplate the injection of the ROS or of the systemicinducer into individual plants or leaves, nor the administration ofgaseous ozone.

If a microbial inducer is to be used, the method is practiced byadministering one or more ROS within about 2 to about 36 hours(preferably, about 12–24 hours) of administration of the microbialinducer of SAR. The microbes are not applied at or about the same timeas the ROS since an ROS such as hydrogen peroxide would tend to kill themicrobes and might decrease the desired effect. It should be noted thatsince kelp and other seaweeds are used in dried form or as liquidextracts, there is no concern that the ROS would kill them. Accordingly,they are generally used following the guidelines for chemical systemicinducers even though they are plants.

An ROS-chemical inducer mixture can be mixed up to a week before use,preferably within 24 hours of intended use, and more preferably within 4hours of intended use. Most preferably, the mixture is made immediatelybefore use, because the ROS tends to decompose once mixed. Typically,the ROS and inducer will be mixed at concentrations where each one canindependently in a range between 1 ppm and about 100,000 ppm. Morepreferably, the range is about 50 ppm to about 50,000 ppm. (Applicationsbelow 100 ppm will typically be used only where longer periods ofapplication are contemplated.) Still more preferably, the range is about100 to about 25,000 ppm. Even more preferably, the range is about 100and about 10,000 ppm.

While the range of either component can vary, in general, it ispreferred that the systemic inducer be applied in a range from aboutequal to about one-twentieth the concentration of the ROS. In morepreferred embodiments, the systemic inducer is applied at concentrationslower than the ROS. In a most preferred embodiment, the systemic induceris present at about one-tenth the concentration of the ROS in ppm.

In the most preferred embodiment, the ROS concentration is between about500 and 5,000 ppm, and the systemic inducer concentration is about 50 toabout 500 ppm, that is, the inducer is applied at about one-tenth theconcentration of the ROS. As noted in the previous section, since theinducer is typically mixed in a 10% solution, an aqueous solution of theinducer can conveniently be mixed in equal volumes with an aqueousmixture of the ROS to achieve the desired ratio between the two.

Generally, we anticipate that cost and other considerations will leadthe practitioner to apply the chemicals at concentrations within thisrange. In some instances, a practitioner may, however, desire to applythe compounds at a higher concentration. There is an upper limit on theconcentration of ROS which can be applied to a plant without toxicity,and the upper limit varies for different types of plants. Citrus plants,for example, can tolerate relatively high levels of ROS. The upper limiton the concentration of the ROS for any particular plant type can beroutinely determined by any of several methods known in the art, such asexposing sample plants of the type in question to various concentrationsof ROS and examining the plants for signs of stress, such as browning oftips of leaves, indicating that the concentration at which the stresssigns occurred is too high for that type of plant. Upper limits on theconcentration of systemic inducer can be determined in the same manner.

It should be noted that with regard to soil applications, thecompositions and methods of the invention are preferably prepared,applied, or both, without exogenously added divalent cations.Additionally, in soil applications, the compositions and methods of theinvention are preferably prepared, applied, or both, without exogenouslyadded cation redox reducing agents. For foliar applications, thepresence of divalent cations or of cation redox reducing agents ispermissible, but the compositions and methods can be prepared or appliedwithout these agents if desired.

IV. Application of the Agents of the Invention

a. Foliar Application

The application of substances to the foliage, or above-ground portionsof plants, is known as foliar application. Foliar application has beenperformed on farms, in greenhouses, on flowers, and in otheragricultural settings for decades, and is performed in any of a varietyof ways known in the art. For example, farmers routinely applypesticides and other agents to their crops by means of tractor mountedsprayers, by crop dusting, through pressurized sprinklers, and throughsystems such as elevated hoses used to spray grapevines.

Typically, the ROS/inducer mixture is dissolved or diluted in water, asappropriate, before use. For foliar application, it is preferred toapply the ROS/inducer mixture at a concentration between 1 ppm and about100,000 ppm. More preferred is a range of about 50 to about 50,000 ppm.Even more preferred is a range of concentration between about 1000 andabout 7000 ppm. Particularly preferred is a range between about 1,750ppm and about 5,000 ppm. In a more preferred embodiment, theconcentration is about 3,500 ppm. Most preferred is a concentrationabout 2,500 ppm. (As noted earlier, all of these concentrations refer tothe ROS component only, with the systemic inducer content, preferably atabout 10% of that of the ROS, being implied. Thus, the most preferredconcentration set forth in this paragraph is the concentration of theROS component, with the presence of about 250 ppm of systemic inducerbeing implied.) We have found that a concentration of about 2,500 ppm ishigh enough to be effective, but provides a margin of safety formathematical errors in applying the invention in practice. Since farmershave been accustomed for years to mixing pesticides, fertilizers, andother agricultural chemicals for use in their fields, the mixing andapplication of an ROS/inducer mixture is well within a farmer's skill.Nonetheless, errors can occur. The preferred concentration of 2,500 ppmis such that if the farmer accidentally halves the concentration (to1,250 ppm), it will still be high enough to be effective, while if it isaccidentally doubled (to 5,000 ppm), it will not be high enough to betoxic to the plants. Lower or higher concentrations can, of course, beemployed, at the user's option, within the limits noted in precedingsections.

The amount of the mixture to be applied to the fields will depend onseveral variables. In foliar application, the goal is to moisten thefoliage. How much water is necessary to accomplish this will dependlargely on the amount of foliage to be covered and the precision of themethod of application in directing the mixture to the foliage withoutalso wetting the surrounding area. The amount of foliage will depend,for example, on the amount of age of the plants (young plants typicallyhave smaller leaves than mature plants), the type of plant (differenttypes of plants differ in the amount and density of their foliage) andthe health of the plants. Farmers have, of course, applied variouschemicals to their crops for years, and are well familiar with judgingthe amount of liquid needed for foliar application on crops of differentages and types. Once the amount of liquid to be used is determined, theamount of ROS/inducer mixture to be added to achieve any desiredconcentration in parts per million is readily determined. Thedetermination of whether the rate of application is sufficient tomoisten the foliage is also easily made and the amount readily adjusteduntil a satisfactory rate is achieved.

It should be noted that some systems, such as sprinkler systems, spraythe whole plant while they water the soil. In the art, and as usedherein, such methods are considered soil applications since theirpurpose is to soak the ground and not merely to wet the leaves or otherportions above the ground.

As a guide to the practitioner, the table below sets forth foliarapplications which were found particularly useful for certain crops infield trials conducted in Mexico. It is anticipated that optimization ofrates of application and volumes of spray water will be necessary forcrops raised under different conditions of temperature and moisture.

TABLE 1 Preferred foliar applications for different crops. Rate- SprayWater Ounces Crop Gallons per Acre /Acre Application Frequency Lettuce,Crucifers,  10–50*  8–16 Apply starting at Asparagus, Garlic,germination at an interval and Cairots of 10 days or less according toconditions.** Tomato, Chile, 10–80  8–26 Apply starting at Melon,Cucumber, germination at an interval Potatoes of 14 days or lessaccording to conditions. Citrus 200–400 64–96 Apply during first rootflush. If necessary repeat in 30 days. Wine, Raisin, &  50–200 16–64Apply every 14 days. Table Grapes Under adverse conditions apply every 7to 10 days. Legend for Table 1. *The spray application depends on theamount of leaf surface area. The first number set forth for each croptype or group ofcrop types is for young plants, the second is for moremature plants with larger leaf areas. The practitioner can determinewhen toswitch to a higher water volume by determining when the lowervolume becomes insufficient to wet the entire leaf surface of the plant.**The “conditions” are the degree of pathogen pressure on the crops. Forexample, if downy mildew is seen on a lettuce crop,the interval beforethe next application will be decreased by a day. If downy mildew isstill observed, the interval for the next applicationwill be decreasedby a day, and so on.

b. Soil Application

In soil application, the soil is preferably first saturated to wet theparticles of the soil so that the ROS/inducer mixture can move freely inthe soil and reach the roots of the plants. Therefore, preferably thesoil is saturated to 70–80% field capacity with ordinary water prior toROS/inducer application. The ROS/inducer mixture is then applied at aconcentration of between 1 and about 100,000 ppm. Typically, theconcentration will be between about 500 ppm and about 10,000 ppm,preferably at a concentration of about 750 ppm to about 7,500 ppm, andmore preferably at a concentration of about 800 ppm to about 5,000 ppm.The particular concentration to be chosen varies primarily according tothe flow rate of water permitted by the method of application. Methodshaving a higher flow rate generally require a lower concentration ofROS/inducer mixture, perhaps because more water containing the mixturereaches the roots of the plants. Conversely, lower flow rates willgenerally require higher concentrations of ROS/inducer mixture.Alternatively, the time of the application of the mixture can bealtered. Thus, use of a low flow rate and low concentration of mixturecan be balanced by increasing the time in which the water containing themixture is applied. Thus, halving the flow rate or concentration ofmixture can be compensated for by doubling the application time of thewater-mixture solution. While flow rate is a particularly importantvariable, the crop to which the mixture is being applied may also helpdetermine the concentration of mixture to be applied. Typically,perennials take higher concentrations than do annuals.

It should be noted that the farmer is usually well aware of the flowrate per acre of the irrigation or other soil application system inplace on his or her property, as well as the acreage to be covered. Thefarmer can calculate the amount of water which will be used in wateringthe land for any particular amount of time (for example, 300 gallons perminute times 50 acres times 30 minutes is 450,000 gallons of water). Thefarmer can then calculate how much ROS/inducer mixture is needed toresult in an application of the desired concentration of the mixture.

The ROS/inducer mixture is applied for a period of time, typicallyranging from about two minutes to about an hour. In some cases, thepractitioner may want to apply the mixture at a lower concentration, butfor a longer period, such as overnight or over several days. Suchapplications are within the purview of the invention, so long as theyresult in increases in PR proteins, PAL, or HPRG, or of diseaseresistance. The time of the application will also vary according to theparticular method employed. For drip systems, the mixture is applied forabout 5 minutes to about 45 minutes. More preferably, the mixture isapplied for about 9 minutes to about 30 minutes. Even more preferably,the mixture is applied for about 15 to about 25 minutes. Consistentlygood results have been achieved in our tests when the mixture is appliedfor about 20 minutes. Accordingly, that period of application is themost preferred.

The practitioner will appreciate that different systems of applicationhave different flow rates. For example, overhead sprinklers generallyhave relatively higher flow rates than do drip systems, and thepreferred application time is correspondingly less: from about 4 minutesto about 10 minutes. Microsprinkler systems such as Fan Jet™ typicallyhave flow rates between that of drip systems and that of sprinklers, andaccordingly have application times somewhat higher than that ofsprinklers, with about 10 to about 15 minutes being preferred.

The ROS/inducer mixtures are typically applied to the soil by being runthrough a hose, pipe, drip, sprinkler, irrigation channel, or othermechanism. In practice, the devices used are not necessarily precisionequipment. Accordingly, when the water flow is turned off, water willtypically continue to drip or run from the hose or through theirrigation channel or other applicator for some time. It is thereforeunderstood that the times of application will generally be anapproximation and will be measured from the start of the flow of themixture to when the flow of the mixture is turned off, whether or notsome of the mixture continues to drip or run from the applicator.

Following application of the ROS/inducer mixture as set forth above, themixture will typically be in the top few inches of soil. For manyplants, the root system is deeper in the soil. It is therefore desirableto help move the mixture 6 to 12 inches into the soil to reach the rootstructures involved in active uptake. To achieve this, it is desirableto use a “water push” to create a concentration gradient afterapplication of the ROS/inducer mixture. This is achieved by followingthe application of the ROS/inducer mixture with an application of water.The water application can be as short as a few minutes or as long asseveral hours. Preferably, however, the water application is betweenabout 30 minutes and about one and one half-hours and more preferably isabout one hour. Such “water pushes” to create concentration gradientsare commonly used by farmers in applying agricultural chemicals and areaccordingly well known in the art.

In soil applications, the ROS/inducer mixture is used without cationredox reducing agents or divalent cations having redox potential inamounts sufficient to reduce the levels of living microorganisms in soilaround the roots of the plants (in combination with the ROS and inducermixture) by 40% or more. The levels of microorganisms in soil can bereadily determined by techniques well known in the art. For example,soil dilution plating assays can be performed by taking a gram of soil,suspending it in 10 mls of sterile water, making a serial dilution andplating each dilution onto a suitable growth medium. This will determineculturable colony counts in the soil sample. In this instance, one wouldperform this assay on two soil samples with similar microorganisms andlevels of those microorganisms and comparing the level of the livingmicroorganisms remaining in the treated sample against the level of theliving microorganisms in the untreated (control) sample.

V. Uses of the Invention

The invention can be used to protect almost any plant capable ofresponding to pathogenic attack with systemic acquired resistance.Assays for determining whether a particular type of plant can benefitfrom the induction of systemic acquired resistance by means of theinvention are well known in the art. For example, northern blots can beperformed to determine whether transcription of genes for PAL, HRGPs,chalcone synthase, peroxidase, or pathogenesis-related proteins havebeen upregulated in response to treatment with a ROS/inducer mixture.Exemplar assays are taught in the Examples.

The plants to be protected by means of the invention can be dicots, suchas carrots, lettuce, tomatoes, grapes, citrus fruits, and beans, ormonocots, such as corn. The plants can be grown for human or animalconsumption, such as grains, vegetables, and fruits, can be intended fordecorative use, such as flowers, or can be intended for ornamental use,such as trees grown for use as Christmas trees or plants intended foruse as house plants. Further, they can be plants grown for fiber, suchas cotton plants, for use as turf, for example on golf courses, lawns orballfields, or for use as or in medicaments. Most commonly, theinvention will be used to protect plants grown in fields as crops or inother open conditions, such as tree farms or turf; the invention can,however, also be used to protect plants grown in settings such asgreenhouses and hothouses.

The invention can be used to protect plants against any pathogen againstwhich systemic acquired resistance can be generated. Pathogens againstwhich SAR can be raised include a variety of bacteria and viruses, anumber of fungi, nematodes, Phylloxera, and even aphids. The Examplesdemonstrate the use of the invention to protect crops in the fieldagainst several pathogens, including insects, fungi and nematodes.

Because the invention protects crops against at least a portion of thedamage which would otherwise be caused by these pests, a higherpercentage of the crops grown for human consumption can be sold as firstquality crops. Moreover, since less of the crop is unmarketable, theinvention results in a higher yield per acre. These factors combine toresult in higher revenues per acre for the farmer.

VI. Methods for Determining if PAL, HPRG, Peroxidase, Chalcone Synthase,Pathogenesis-Related Proteins, or Their Transcripts are Increased

A number of methods are available to determine if PAL, HPRG, peroxidase,chalcone synthase, pathogenesis-related proteins, or their transcripts,or other enzymes or proteins of interest are increased. One can, forexample, detect an increase in RNA levels in response to a ROS/inducermixture in comparison to a control by means of assays such as northernblots. Exemplar northern blot assays are discussed in the Examples,below. Alternatively, a protein, such as a HRGP, can be used to raiseantibodies against the protein by injecting it into mice or rabbitsfollowing standard protocols, such as those taught in Harlow and Lane,Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1988). The antibodies so raised can then be used todetect the presence and amount of the protein in a variety ofimmunological assays, such as ELISAs, fluorescent immunoassays, andWestern blots.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily-recognize a variety ofnoncritical parameters which could be changed or modified to yieldessentially similar results.

Example 1 Preparation of ROS and Systemic Inducer and Creating anROS/Inducer Mixture

This example demonstrates the making of preferred forms of ROS andsystemic inducer for forming compositions of the invention and for usein the methods of the invention. In the procedures set forth, normalprotocols for preparing the ROS and the systemic inducer have beenaltered to include amounts of chelating agents, sequestering agents, andsurfactants our work has indicated render the final ROS/inducer mixturethe most effective.

Making peracetic acid (“PAA”) for use in the invention commences with adecision as to the amount of PAA desired, for example, 1 gallon or 100gallons. The PAA is then made by blending the following ingredients, inthe order shown. We prefer to use a 5% solution of PAA. At a 5%solution, PAA weighs 9.4 pounds per gallon, and it is therefore possibleto calculate the weight of any desired amount of PAA (for example, 10gallons would equal 94 pounds). The percentages below denote the weightof the ingredient to add, in percent of the final weight of the finalamount of PAA desired. Following the recipe below, for example, to mixten gallons of PAA, one would use 47 pounds (50% of 94) of 50% hydrogenperoxide. The recipe is as follows:

 25% De-ionized water  50% 50% Hydrogen peroxide  11% Glacial aceticacid .05% Tetrapotassium pyrophosphate .05% Versonex 80 ™ (a chelatingagent; other commercially available chelating agents can be substituted)This mix is then brought up to final weight with 13.9% de-ionized water.Similar calculations can be made to mix solutions of PAA at differentconcentrations, if desired.

For making a solution of salicylic acid, a preferred inducer of systemicresistance, the following means is preferred. As in the method above,one first decides the total amount of the product desired and calculatesthe weight of that amount. To make the 10% solution of salicylic acidand humic acid which is the preferred embodiment (a 10% solution ofsalicylic acid weighs 9.5 pounds per gallon), the ingredients are mixedaccording to the following recipe, by percentage of the final weight ofthe mixture:

 30% De-ionized water  10% Salicylic acid  10% Humic acid (in the formof a 12% aqueous solution)  10% Caustic potash   1% 80% Phosphoric acid2.5% Tergitol 15 S 9 ™ 2.5% Triton H-66 ™   1% Versonex 80 ™  12%Tetrapotassium pyrophosphateThis mix is then brought up to final weight with 21% deionized water. Asnoted, this recipe results in a 10% solution of systemic inducer. Thesolution can then be mixed in an equal volume with an ROS, such as the5% peracetic mixture described above, to obtain a mixture withconcentrations in the preferred ratio of 10:1 ROS to systemic inducer.In the Examples below, humic acid in an aqueous solution was substitutedfor one quarter of the deionized water so that it constituted 10% of thefinal weight of the salicylic acid solution.

The Examples below include laboratory and field trials of the invention.In the laboratory trials, some plants were contacted with ROS mixturewithout also being contacted with systemic inducer mixture. Where thesystemic inducer mixture was added, it was added at 200 ppm. Referencesto concentrations of the inducer refer to the concentration of themixture, with the surfactants and other agents noted above.

In the field trials, the concentrations stated are of the ROS portion ofthe mixture. In each field trial, an equal volume of the systemicinducer mixture was added so that the inducer mixture was present atabout one-tenth the concentration of the ROS mixture.

Example 2 Induction of Expression of the PR-1a Gene

This example shows that plants sprayed with an ROS/inducer mixture in alaboratory showed greater expression of the gene encoding thepathogenesis-related protein PR-1a than did plants sprayed with eitheran ROS alone or with a systemic inducer alone.

Fourteen-day old red kidney bean plants (Phaseolus vulgaris) weresprayed from an overhead boom until the leaves were wet and spray beganto run off them. Plants were treated with one of the followingtreatments: water, the ROS, peracetic acid, at a concentration of 3,500ppm, peracetic acid at a concentration of 10,000 ppm, a ROS/inducermixture, at 3,500 ppm of the ROS and 200 ppm of the inducer (salicylicacid) mixture, of the same mixture at 10,000 ppm for the ROS and 200 ppmof the inducer, and of just the inducer, at 200 ppm.

The plants were then left for 24 hours, after which the leaves weresubjected to extraction of their total RNA by the procedure described inLogemann, J., et al, “Improved method for the isolation of RNA fromplant tissues.” Anal Biochem. 163:16–20 (1987). Formaldehyde gels werethen prepared, loaded with 10 μg of the total RNA, and run to separateRNA by size, according to standard methods (Sambrook, J., et al.,Molecular Cloning, A Laboratory Manual. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2nd ed. 1989)). The gels were thenblotted (as described in Sambrook, supra), and hybridized to a probe forPR-1a prepared according to the method described in Zdor, R., and A. J.Anderson, “Influence of root colonizing pseudomonads on defensemechanisms of bean.” Plant and Soil. 140:99–107 (1992). Nonradioactiveprobe was prepared via random priming of only the cloned insert.Hybridization was observed and analyzed by chemiluminescent detection ofthe bound probes using the “Genius System,” (Boehringer MannheimCorporation, Indianapolis, Ind.), following the manufacturer'sdirections. Equal loading of the RNA was judged by ethidium bromidestaining of the ribosomal RNA bands.

The results are shown in FIG. 1, which is a northern blot of RNA probedfor PR-1a. While no water-only control was run in this northern, ourprevious work has demonstrated that no RNA message is detectable forplants treated only with water. PR-1a gene RNA expression increased upontreatment with 3,500 ppm of peracetic acid (lane 1) and higher amountsare seen at treatment with 10,000 ppm of peracetic acid (lane 2). Thesystemic inducer mixture, at 200 ppm, also showed increases of RNAaccumulation (lane 5). The ROS/inducer mixture treatment, at both 3,500ppm of ROS and 200 ppm of salicylic acid (lane 3) and of 10,000 ppm ofROS and 200 ppm of inducer mixture (lane 4), showed dramatic,synergistic effect, reflecting an effect clearly greater than a simpleadditive effect of the increase in RNA transcript accumulation caused byeither of the two agents alone.

Example 3 Induction of the Gene Encoding Phenylalanine Ammonia Lyase

This example shows that the use of a ROS/inducer mixture causes anincrease in transcription of a gene encoding phenylalanine ammonia lyase(“PAL”), another marker for plant resistance.

Bean plants were grown and treated in the same manner as in the previousExample, except that a water-only control was run, but not a test ofsystemic inducer without PAA. Total RNA was extracted and gels wereloaded, run, and analyzed following the procedures discussed in previousExample, with the exception that no lane was run containing RNA fromplants sprayed with a systemic inducer without an ROS present. Probe forPAL mRNA transcripts was prepared following the procedures set forth inBlee, K. A., and Anderson, A. J. “Defense-related transcriptaccumulation in Phaseolus vulgaris L. colonized by the arbuscularmycorrhizal fungus Glomus intraradices Schenck & Smith.” Plant Physiol.110: 675–688 (1996).

The northern blot resulting from this study is set forth as FIG. 2. Nohybridizing RNA can be seen in the lane for the RNA from thewater-treated controls (lane 1). The lanes for treatment with peraceticacid at 3,500 ppm (lane 2) and at 10,000 ppm (lane 3) show an increasein mRNA for PAL genes over that of the water control, with the lane for10,000 ppm treatment showing a substantial increase in the amount of RNAcompared to the lane for treatment with 3,500 ppm of PAA. The fourthlane shows that treatment with 3,500 ppm of the ROS peracetic acid incombination with an inducer mixture (at 200 ppm) resulted in levels ofPAL induction at least equal to that of application of 10,000 ppm ofperacetic acid in the absence of an inducer.

Example 4 Induction of Gene(s) Encoding Hydroxyproline-Rich Glycoproteinby an ROS/Inducer Mixture

Hydroxyproline-rich glycoproteins (“HPRG” or “HYP”) are known to bedeposited in increased amounts in plant cell walls when plants arechallenged by pathogens, and are thought to strengthen the cell walls.This Example shows that there is an increase in the level of RNA for HYPupon treatment with a ROS/inducer mixture, and that the increase waslarger than that seen upon treatment with a ROS alone or with a systemicinducer alone.

Bean plants were grown and treated as in Example 2. Total RNA wasextracted and gels were loaded, run, and analyzed following theprocedures discussed in Example 2. Probes for HYP were preparedfollowing the procedures set forth in Blee and Anderson, supra.

The northern blot resulting from this study is set forth as FIG. 3. Thelanes for treatment with peracetic acid at 3,500 ppm (lane 2) and 10,000ppm (lane 3) show an increase in RNA levels from HYP genes over that ofthe water control (lane 1), with the lane for treatment with 10,000 ppmshowing substantially more hybridizing RNA than does the lane fortreatment with 3,500 ppm (compare lane 3 to lane 2). Lane 6, which showsprobing of RNA from plants treated with a systemic inducer (salicylicacid, at 200 ppm) but without an ROS present, shows a level of HYPinduction somewhat greater than that of the lane reflecting treatmentwith an ROS at 3,500 ppm (lane 2) but less than that of the lanereflecting treatment with an ROS at 10,000 ppm (lane 3). Lane 4,containing RNA from plants treated with both an ROS at 3,500 ppm and asystemic inducer mixture (according to the recipe set forth inExample 1) at 200 ppm, shows a much greater level of RNA induction thanis true for plants treated only with 3,500 ppm of the ROS (lane 2), andindeed is greater than the induction seen for 10,000 ppm of ROS alone(lane 3). Lane 5, reflecting treatment with an ROS at 10,000 and thesystemic inducer mixture (at 200 ppm) also shows a very strong inductionof HYP mRNA in comparison to the lanes reflecting application of an ROSat the same concentration (lane 5) or of the systemic inducer mixture(at 200 ppm) alone (lane 6).

Example 5 Field Trial of ROS/Inducer Mixture to Treat NematodeInfestation in Table Grapes

Field trials were designed by University of California extension agents,who were instructed to design them to the same standards as forUniversity of California experiments. This trial shows use of theinvention to treat a nematode infestation in table grapes. Nematodespresent in high concentrations were Ring, Citrus (Tylenchulus), Dagger(Xiphinema), Stubby Root (Trichodorus), and Lesion (Pratylenchus).

A ROS/inducer mixture of peracetic acid and salicylic acid, withsurfactants, sequestering agents, and caustic potash present in smallamounts (in this and in the remaining examples referred to as a“PAA/inducer mixture”), as described above, was applied to a crop oftable grapes. Three applications were made, starting early in thegrowing season. For the first application, 1 gallon per acre of the PAAand 1 gallon per acre of the 10% inducer mixture were applied, followedby a second application 21 days later at a rate of ½ gallon of each peracre, followed by a third application 21 days after that, at the samerate as the second. Application was in water, made by drip irrigationfor 30 minutes, at a flow rate of 9 gallons per acre per minute, to afinal concentration of 1900 ppm, followed by a “water push.” The trialwas randomized, with six replicates per treatment, and 140 samples weretaken from each replicate. The control was treatment with water only.

Results: Of the fields treated with water only, the yield (in number of21 pound boxes of fruit) was: 373 boxes of No. 1 quality grapes and 282boxes of No. 2 quality grapes, for a total yield of 656 boxes. Fieldstreated with the ROS/inducer mixture yielded 414 boxes of No. 1 fruitand 373 boxes of No. 2 fruit, for a total of 787 boxes. At $10 per box,the difference in yield increased revenue by $1310 per acre.

Example 6 Field Trial of the Invention Testing Ability to Reduce PowderyMildew on Table Grapes

This Example shows the ability of the invention to reduce the percentageof table grapes infected with powdery mildew, a fungal infection.

In this Example, the plants were examined to determine the number withLevel 1 mildew (fewer than 3 grapes per plant with active mildew) beforeand post-treatment with a PAA/inducer mixture. A 50 acre vineyard wasdivided into 16 replicates of 88 vines each, for a total of 1408 vinesin the trial. The vines in each replicate were examined, and thepercentage with Level 1 powdery mildew determined. The field was thentreated in mid-summer with a single, foliar application of PAA/inducermixture by an air blast sprayer at a concentration of 1250 ppm, appliedas 1 quart of mixture in 200 gallons of water. The replicates were thenexamined after 1 week and the percentage with Level 1 mildew determined.

The results are shown in FIG. 4. For every replicate, the percentage ofplants with Level 1 mildew was markedly lower than the percentage ofplants with mildew for the same replicate prior to treatment.

Example 7 Two Field Trials of the Invention Testing Ability to ReducePythium and Nematode Damage to Carrots

This Example sets forth two field trials which together show the abilityof the method of the invention to increase yield of carrots and toreduce the amount of Pythium and nematode damage to carrots compared toone of the most widely used conventional agents. Pythium, a fungus whichattacks through the root system, and nematodes, which strike at thegrowing tip of the carrot during the first 20 days of growth, cause thecarrot to split into a fork around the damaged tissue. Carrots sodamaged cannot be sold as first quality, i.e., fresh carrots for eating,for which the farmer receives the highest price.

a. Field Trial A

This trial was conducted to determine yield information. Tests were ofside-by-side comparisons of two 50-acre plots, with six replicates each.Carrots being treated with the PAA/inducer mixture were given a “germwater” application (in which a seed planted in fairly dry soil is givenits first watering, to encourage its germination, with the treatedwater) by irrigation with a pressurized sprinkler system at anapplication rate of 1.25 gallons of mixture per acre, for aconcentration of 1100 ppm. The carrots were then treated with twoadditional applications, one ten days after the first application andanother ten days after that, at a rate of 1 gallon per acre.

To test the efficacy of the invention against currently usedconventional agents, a second group of carrots were grown using awidely-used soil fumigant, Vapam™ (generically, metam-sodium), tocontrol pests. This agent is applied to the soil as a liquid, which thenemits carbon disulfide gas. Since the gas kills most living things withwhich it comes in contact in high concentrations, including growingplants, it is applied before the field is planted. It was applied inaccordance with the label directions.

Results: Of the carrots treated with the ROS/inducer mixture, 98.2% ofthe carrots treated with the ROS/inducer mixture were marketable, ascompared to 96% of the carrots grown after treatment with Vapam™. TheROS/inducer mixture also resulted in a yield of marketable carrots, 29.1tons/acre, almost a ton per acre higher than did the fields treated withVapam™, which produced 28.2 ton/acre. Thus, the method of the inventionresulted in slightly better quality, and an increased yield, compared toa widely-used, but highly toxic, conventional pesticide.

b. Field Trial B

This trial was intended to measure the yield of “cello packs” ofcarrots, 50-pound plastic bags of carrots with their tops removed.“Cello pack” carrots command one of the highest prices the farmer canreceive for carrots; it is advantageous to have carrots with a goodweight and diameter so that fewer carrots are needed to fill each50-pound bag. The trial was set up and conducted as for Field Trial A,above, including the size of the fields, the dates of application, andthe concentrations of the agents applied. Carrots intended for cellopack, however, remain in the ground for an additional 10 days to allowthem to gain more thickness and weight; this was done for each group inthis trial.

Results: Pythium damaged 12.7% of the crop in fields treated with Vapam™compared to almost zero (0.1%) in fields treated with the ROS/inducermixture. Nematodes damaged 11.9% of the crop in fields treated withVapam™ compared to 2.9% in fields treated with the ROS/inducer mixture.Accordingly, in replicate plots, the ROS/inducer markedly reduced damagefrom Pythium and nematodes compared to this widely used, but highlytoxic, conventional pesticide.

The size of the carrots also differed between the two treatment groups.Nine percent more of the carrots grown in the fields treated with theROS/inducer mixture had a diameter in the desirable ½ to 1 inch rangecompared to the carrots treated with Vapam™ (81% to 74%), while thepercentage with diameters smaller than ½ inch in the ROS/inducer treatedgroup was 4% smaller than that of the Vapam™ treated crop (19% to 23%).The ROS/inducer treated crop did have a smaller percentage of carrotswith diameters over 1 inch compared to the Vapam™ treated crop (0% to3%).

More importantly, the yield differed between the two treatment groups.Fields treated with the ROS/inducer mixture yielded 29 tons/acre,compared to 22.7 tons/acre produced by fields treated with Vapam™, adifference of almost 29%. Even more importantly, this difference inyield per acre was reflected in an almost 48% difference in the numberof cello packs of carrots packed per acre, with an average of 177 bagsper acre for the fields treated with the ROS/inducer mixture, and anaverage of 120 bags per acre for the fields treated with Vapam™. Thus,the method of the invention resulted in a markedly increased productionof high value crop, compared to a widely-used, but highly toxic,conventional pesticide.

Example 8 Field Trial of the Invention to Determine Ability to ReduceViral Damage to Tomatoes

This trial shows the effect on the method of the invention on reducingdamage to tomatoes from viral infections.

The trial plot comprised 84,000 square feet divided into 24 side-by-sidereplicates of treated and nontreated plants. Seventeen thousand fourhundred plants were involved in the trial. Viruses present in the fieldwere identified as: cucumber mosaic, alfalfa mosaic, and curly top. Inaddition, plants showed three different sets of symptoms which appearedto be due to viruses, but which could not be attributed to a specificvirus.

Results: Treated plants showed an average of a 60.4% reduction in theincidence of virus symptoms compared to non-treated replicates.

Example 9 Field Trial of the Invention to Determine Ability to ReduceDowny Mildew Damage to Lettuce

This trial shows the effect on the method of the invention on reducingdamage to lettuce from downy mildew. Downy mildew is a fungal infectionwhich causes lettuce to rot after harvest and is the greatest singleproblem in agricultural production of lettuce. It requires frequenttreatment to keep it under control.

Plants treated with the PAA/inducer mixture were given foliarapplications by overhead sprayer each at a rate of 16 oz. in 50 gallonsof water per acre, in five applications each of which was spaced tendays apart. Control plants were given a combination of two conventionalfungicides, Alliette™ and mancozeb, currently in wide use forcontrolling downy mildew in lettuce. The fungicides were also appliedevery ten days, following label directions. The plants were thencompared for the number of leaves infected with downy mildew.

The results for a total of six replicates showed that, for the fieldstreated with the PAA/inducer mixture, a total of 45 plants were infectedwith downy mildew, and 704 were not, for an average infection rate of6.1%. For plants treated with the conventional fungicides, a total of117 plants were found to be infected out of 842 examined, for an averagerate of infection of 12.2%. Thus, in this trial, treatment with aPAA/inducer mixture resulted in halving the infection of the plants bydowny mildew.

Example 10 Two Field Trials of the Invention to Determine its Ability toReduce Red Scale on Citrus Fruits

These trials show the effect on the method of the invention on reducingdamage to oranges from the insect pest red scale. The presence of redscale on the fruit renders it unmarketable for sale as fresh fruit andrequires that the crop be sold at lower prices for use in juice. Theinsects also cause significant damage to the trees themselves.Evaluation of the number of insects on the leaves is a measure todetermine the level of infestation on the trees before the insects reachthe fruit.

a. Field Trial A

An orange grove was divided into two groups of trees, one to be treatedwith PAA/inducer and one to receive only an equivalent application ofplain water. Trees to be treated with the PAA/inducer mixture were givena foliar application by sprayer, at a concentration of 2500 ppm, in 200gallons of water per acre, a few weeks before harvest; and samples weretaken for evaluation two weeks later. Five acres were treated Threereplicates were taken for each group of trees. Each replicate was madeup of 10 leaves sampled from 5 trees, for a total of 50 leaves. Leavesselected for the sample were pulled from both the inside and the outsideof the canopy of the trees. Insects present in addition to red scalewere aphytis, predator mite, and lacewing; the numbers below, however,represent the numbers of red scale only.

Results: Among treated trees, 1173 live red scale insects were countedand 4833 dead red scale insects were counted, for a 20% to 80% (or 1:4)ratio of live to dead insects. Mortality was especially high among theearliest stages of the insect's life cycle: for example, only 5% of thecrawlers were alive. Among control trees, treated only with water, 1637live red scale insects were counted and 275 dead red scale insects werecounted, for an 86% to 14% ratio. Ninety-six percent of the crawlerswere alive. Thus, the application of the PAA/inducer mixture reversedthe percentage of live to dead insects compared to the controls. Table 2shows the percentages of dead insects by life stage for treated treescompared to untreated trees.

TABLE 2 PAA/SA Treated Untreated Life Stage % Dead % Dead Crawlers 95% 4% White Cap 88% 11% Nipple/2nd Molt 77% 11% Female: 64%  6% Male: 82%39%

b. Field Trial B

A second trial was run with the same application dates, concentrations,controls and sampling and evaluation criteria as reported for FieldTrial A, above.

Results: As shown in Table 3, a high percentage of the insects found onthe treated plants were dead in every life stage, with higher mortalityamong the earlier life stages. The untreated trees had a lowerpercentage of dead insects (and, hence, a larger percentage of liveinsects) at every life stage, with, for example, less than one fourththe percentage of dead insects in the nipple/2nd molt stage compared tothe treated trees.

TABLE 3 PAA/SA Treated Untreated Life Stage % Dead % Dead Crawlers 97%52% White Cap 95% 35% Nipple/2nd Molt 85% 13% Female: 71% 15% Male: 86%47%

Example 11 Two Field Trials of the Invention to Determine Ability toReduce Phylloxera and Nematode Infestation of Wine Grapes

These trials show the effect on the method of the invention on reducingdamage to two different varieties of wine grapes from phylloxera andfrom nematodes. Phylloxera is a root louse that deposits eggs in thesoil; when they hatch, the offspring parasitize the plants.

a. Field Trial A

This trial was a phylloxera trial on grapes of the varietal Chardonnay.A one hundred-acre vineyard of this grape variety was divided into 2fifty-acre plots. One portion of the vineyard was then treated with thePAA/inducer mixture a total of four times, by soil applications spaced28 days apart. Each application was at a rate of ½ gallon of mixture peracre, to provide a concentration of 1900 ppm for 30 minutes. The otherportion of the vineyard was treated with Enzone™, a pesticide commonlyused to treat pholloxera infestation, following label directions.

Results: The PAA/inducer treated acres produced an average of 5.4 tonsof grapes per acre. The nematicide-treated acres produced an average of4.2 tons per acre. Thus, the acres treated according to the inventionyielded approximately 28.5% more grapes per acres (in weight) than didcomparable acres treated with a conventional agent.

b. Field Trial B

This trial was a nematode control trial on grapes of the varietalZinfandel. The plots were selected and divided as in Field Trial A,above. The plots treated with the PAA/inducer mixture received the sametreatment (that is, the same quantities and concentration of mixture, onthe same dates) as did the plots described in Field Trial A of thisExample, above. The control plots were treated with a conventionalnematicide, DiTera™.

Results: The PAA/inducer treated acres produced an average of 4.52 tonsof grapes per acre. The nematicide-treated acres produced an average of3.49 tons per acre. Thus, the acres treated according to the inventionyielded approximately 29.5% more grapes per acres (in weight) than didcomparable acres treated with a conventional nematicide

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of increasing yield of plants, comprising contacting foliageof the plants with an effective amount of (i) a plant systemic inducerand (ii) a reactive oxygen species selected from the group consisting ofperacetic acid, hydrogen peroxide, a hydroperoxide, a peroxide, calciumperoxide, potassium percarbonate, and urea peroxide.
 2. A method ofclaim 1, further wherein the plant is contacted with humic acid.
 3. Amethod of claim 1, wherein the systemic inducer is derived from a kelpor other seaweed.
 4. A method of claim 1, wherein the reactive oxygenspecies is selected from the group consisting of: peracetic acid,hydrogen peroxide, calcium peroxide, potassium percarbonate, sodiumpercarbonate, and urea peroxide.
 5. A method of claim 1, wherein theplant systemic inducer is selected from the group consisting of:salicylic acid, jasmonic acid, isonicotinic acid, arachidonic acid,phosphorus acid, dichloroisonicotinic acid, and benzothiadiazole.
 6. Amethod of claim 1, wherein the plant systemic inducer is a microbenonpathogenic to the plant.
 7. A method of claim 1, wherein the plantsystemic inducer is salicylic acid.
 8. A method of claim 1, wherein theplant systemic inducer is humic acid.
 9. A method of claim 1, whereinthe reactive oxygen species is peracetic acid.
 10. A method of claim 1,wherein the plant systemic inducer species and the reactive oxygenspecies are mixed together before they contact the plants.
 11. A methodof claim 1, wherein the plants are dicotyledons.
 12. A method of claim1, wherein the plants are of a species edible by humans.
 13. A method ofclaim 1, wherein the plants are selected from the group consisting of:grapes and carrots.
 14. A method for increasing yield of plants,comprising contacting roots of said plants with (i) a plant systemicinducer and (ii) a reactive oxygen species selected from the groupconsisting of peracetic acid, hydrogen peroxide, a hydroperoxide, aperoxide, calcium peroxide, potassium percarbonate, calcium hydroxide,and urea peroxide, wherein the amount of said plant systemic inducer andthe amount of said reactive oxygen species is sufficient to increase theyield of said plants, provided that the plants are not also contactedwith an agent selected from the group comprising a cation redox reducingagent and a divalent cation having redox potential in an amount toreduce levels of microorganisms in soil around the roots by 40% or more.15. A method of claim 14 further comprising contacting said plants withan agent that chelates heavy metal ions or that sequesters heavy metalions from the reactive oxygen species.
 16. A method of claim 14 furthercomprising contacting said plant with a surfactant.
 17. A method ofclaim 14, further comprising contacting said plant with humic acid. 18.A method of claim 14, wherein the reactive oxygen species is selectedfrom the group consisting of peracetic acid, hydrogen peroxide, calciumperoxide, calcium hydroxide, potassium percarbonate, sodiumpercarbonate, calcium hydroxide, and urea peroxide.
 19. A method ofclaim 14, wherein the reactive oxygen species is peracetic acid.
 20. Amethod of claim 14, wherein the plant systemic inducer is selected fromthe group consisting of salicylic acid, jasmonic acid, isonicotinicacid, arachidonic acid, phosphorus acid, dichloroisonicotinic acid, andbenzothiadiazole.
 21. A method of claim 14, wherein the plant systemicinducer is salicylic acid.
 22. A method of claim 14, wherein the plantsystemic inducer is salicylic acid and the reactive oxygen species isperacetic acid.
 23. A method of claim 15, wherein the agent thatchelates heavy metal ions or that sequesters heavy metal ions from thereactive oxygen species is tetrapotassium pyrophosphate.